The Trypanosoma brucei polo-like kinase homologue is an essential morphogenic regulator of the parasite's cytoskeleton. A series of proteomic screens identifies potential TbPLK binding partners and substrates and better illustrates how the kinase functions, yielding novel proteins involved in flagellar positioning.

SummaryPolo-like kinases play an important role in a variety of mitotic events in mammalian cells, ranging from centriole separation and chromosome congression to abscission. To fulfill these roles, Polo-like kinase homologs move to different cellular locations as the cell cycle progresses, starting at the centrosome, progressing to the spindle poles and then the midbody. In the protist parasite Trypanosoma brucei, the single polo-like kinase homolog T. brucei PLK (TbPLK) is essential for cytokinesis and is necessary for the correct duplication of a centrin-containing cytoskeletal structure known as the bilobe. We show that TbPLK has a dynamic localization pattern during the cell cycle. The kinase localizes to the basal body, which nucleates the flagellum, and then successively localizes to a series of cytoskeletal structures that regulate the position and attachment of the flagellum to the cell body. The kinase localizes to each of these structures as they are duplicating. TbPLK associates with a specialized set of microtubules, known as the microtubule quartet, which might transport the kinase during its migration. Depletion of TbPLK causes defects in basal body segregation and blocks the duplication of the regulators that position the flagellum, suggesting that its presence on these structures might be necessary for their proper biogenesis. TbPLK migrates throughout the cell in T. brucei, but the specific locations to which it targets and its functions are geared towards the inheritance of a properly positioned and attached flagellum.

The Polo-like kinase homologue in Trypanosoma brucei (TbPLK) regulates the assembly of a series of organelles necessary for positioning the parasite's flagellum. An analogue-sensitive strategy is used to acutely and specifically inhibit the kinase with a small molecule, making it possible to identify novel TbPLK functions.

The interplay between dendritic cells (DC) and ␥␦ ⌻ lymphocytes represents a network of paracrine and cell contact interactions important for an integrated immune response to pathogens. HIV-1 infection dramatically affects the number and functions of both cell populations, and DC/␥␦ ⌻ cell cross talk may represent a target of virus-induced immune escape. We investigated whether HIV-exposed DC could deliver aberrant signals to interacting ␥␦ ⌻ cells. Here we report that the interaction of human ␥␦ T lymphocytes with HIV-1-exposed autologous monocyte-derived DC, but not direct exposure to the virus, impairs lymphocyte expansion and gamma interferon (IFN-␥) production in response to phosphoantigens. This effect is independent of virus strain and occurred in 55% of the donors analyzed. The donor-dependent variation observed relies on the responsiveness of DC to HIV-1 and is strictly related to the capacity of the virus to suppress the maturation-induced expression of interleukin 12 (IL-12). In fact, ␥␦ T cell response to phosphoantigens is almost completely recovered when this cytokine is exogenously added to the DC/lymphocyte cocultures. Interestingly, we show that ␥␦ T lymphocytes are recruited by HIV-1-exposed DC through a CCR5-mediated mechanism and exert a CCL4-mediated control on virus dissemination within DC and susceptible CD4 ؉ T lymphocytes. These results demonstrate an association between HIV-induced DC dysfunction and alterations of ␥␦ T cell responses. The aberrant cross talk between these two cell populations may contribute to the pathogenesis of HIV infection by further reducing the strength of antiviral immune response. IMPORTANCEThis study provides new evidence on the mechanisms exploited by HIV-1 to evade the host immune response. We report that HIV-1 impairs the cross talk between DC and ␥␦ T lymphocytes, by reducing the capacity of DC to promote functional ␥␦ T cell activation. Interestingly, the virus does not per se interfere with ␥␦ T cell activation, thus highlighting the key role of early DC-HIV-1 interaction in this phenomenon. Furthermore, the results obtained unravel the novel role of ␥␦ T cells in controlling HIV-1 dissemination within the DC population as well as virus transfer to susceptible CD4 ؉ T lymphocytes. The interactions of DC with innate lymphocytes represent a major control mechanism for an integrated immune response to infection. Understanding how HIV-1 harnesses these pathways may provide important insights on the pathogenesis of disease and offer new opportunities for therapeutic interventions.

Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (rhomboid-like protein 4) is an endoplasmic reticulum (ER)–resident protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre–T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys 63 -linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

CLRs are predominantly expressed in macrophages and myeloid DCs, where they play a key role in antigen recognition, scavenging, and host defense against pathogens. To identify novel immunoregulatory cytokines and networks involved in the control of these functions, we analyzed the coordinate effects of IFN-β and IL-3 on CLR expression, antigen uptake, and phagocytosis in human MDMs and MDDCs. We report that these cytokines exert opposite regulatory effects on the expression of CLRs and endocytic/phagocytic activities of MDMs. Specifically, IFN-β markedly inhibits the expression of MR and Dectin-1 during MDM differentiation and impairs the capacity of MDM to internalize antigens and phagocytose unopsonized Candida albicans. Conversely, IL-3 up-modulates MR, Dectin-1, and DC-SIGN, thus allowing more efficient uptake/phagocytosis. Interestingly, IL-3 counteracts the IFN-β effect on antigen uptake/processing by fully restoring MR expression in IFN-β-primed MDMs. In contrast, the phagocytic activity is only partially restored as a result of the failure of IL-3 in counteracting IFN-β-induced Dectin-1 suppression. Notably, IFN-β-mediated impairment of CLR expression/function occurs in macrophages but not in MDDCs. These results identify IFN-β and IL-3 as unrecognized regulators of CLR expression and function, unraveling a novel interaction between these cytokines instrumental for the regulation of the macrophage response to pathogens.

Nanoindentation, laser ablation inductively coupled plasma mass spectroscopy and weighing ion-spiked organic matrix standards revealed structure-property relations in the microscopic jaw structures of a cosmopolitan bristle worm, Platynereis dumerilii. Hardness and elasticity values in the jaws’ tip region, exceeding those in the center region, can be traced back to more metal and halogen ions built into the structural protein matrix. Still, structure size appears as an even more relevant factor governing the hardness values measured on bristle worm jaws across the genera Platynereis, Glycera and Nereis. The square of the hardness scales with the inverse of the indentation depth, indicating a Nix-Gao size effect as known for crystalline metals. The limit hardness for the indentation depth going to infinity, amounting to 0.53 GPa, appears to be an invariant material property of the ion-spiked structural proteins likely used by all types of bristle worms. Such a metal-like biogenic material is a major source of bio-inspiration.

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